Mechanism of Metformin Action in Normal and PCOS Theca Cells

描述（由申请人提供）：双胍类药物二甲双胍的使用是2型糖尿病年轻女性的标准用药，并且在治疗多囊卵巢综合征（PCOS）女性的高雄激素和无排卵症中越来越普遍。体外研究表明，二甲双胍对抑制卵巢鞘雄激素生物合成有直接作用，这对两组女性都有临床意义。然而，二甲双胍抑制卵巢雄激素生物合成的机制目前尚不清楚。我们的初步研究表明，二甲双胍治疗对抑制正常周期和多囊卵巢综合征女性的卵泡细胞繁殖的雄激素生物合成有不同的影响。对经典的二甲双胍激活LKB1/ amp激活蛋白激酶（AMPK）信号级联的研究提供了新的发现，LKB1磷酸化以及AMPK活性在PCOS膜细胞中降低。二甲双胍治疗纠正了多囊卵巢综合征的这些信号缺陷，同时抑制了雄激素的生物合成。在本提案中，我们将验证PCOS卵巢中LKB1/AMPK信号缺陷导致雄激素生物合成增加和细胞生长中断的假设。在Specific Aim 1中，我们将研究二甲双胍激活LKB1/AMPK信号通路抑制PCOS膜细胞雄激素生物合成的潜在机制。我们将研究LKB1、AMPK和盐诱导激酶SIK1和SIK2在二甲双胍抑制正常和多囊卵巢综合征卵泡膜细胞中CYP17和CYP11A1基因表达和雄激素生物合成中的作用。多囊卵巢的病理特征是卵巢有多个小的生长受阻的卵泡。然而，没有报道比较正常和多囊卵巢综合征的卵泡膜细胞的细胞生长或细胞周期控制。我们已经确定了正常和多囊卵巢卵巢膜细胞的细胞周期调节的差异，并观察到二甲双胍治疗多囊卵巢卵巢膜细胞可以改变多囊卵巢卵巢膜细胞和细胞周期进展到正常表型。在Specific Aim 2中，我们将鉴定多囊卵巢综合征（PCOS）卵泡膜细胞的细胞生长和细胞周期缺陷，并探索二甲双胍纠正这些缺陷的信号机制。在正常生理和高胰岛素条件下，LKB1、AMPK和SIK1/2在二甲双胍依赖性细胞生长和细胞周期进展中的作用将被评估。我们相信，明确二甲双胍信号介导PCOS膜细胞雄激素生物合成抑制和细胞生长的机制，将有助于阐明PCOS的病理生理，并为开发新的PCOS治疗靶点提供见解。

DESCRIPTION (provided by applicant): The use of the biguanide drug, metformin, is standard for young women with type 2 diabetes and is becoming prevalent for the treatment of hyperandrogenism and anovulation in women with polycystic ovary syndrome (PCOS). In vitro studies have shown that metformin has direct effects on inhibiting ovarian thecal androgen biosynthesis, which is of clinical relevance to both groups of women. However, the mechanism(s) by which metformin inhibits ovarian androgen biosynthesis is presently unclear. Our preliminary studies suggest that metformin treatment has differential effects on inhibiting androgen biosynthesis in theca cells propagated from normal cycling and PCOS women. An examination of the classical metformin activated LKB1/ AMP-activated protein kinase (AMPK) signaling cascade, provided the novel findings that LKB1 phosphorylation, as well as AMPK activity are decreased in PCOS theca cells. Metformin treatment corrected these signaling defects in PCOS theca cells, while inhibiting androgen biosynthesis. In this proposal we will test the hypothesis that defects in LKB1/AMPK signaling in PCOS theca cells contribute to increased androgen biosynthesis and disrupted cell growth in the PCOS ovary. In Specific Aim 1, we will investigate the underlying mechanism(s) by which metformin activates the LKB1/AMPK signaling pathway(s) to inhibit androgen biosynthesis in PCOS theca cells. We will examine the role(s) of LKB1, AMPK, and the salt inducible kinases, SIK1 and SIK2, in metformin inhibition of CYP17 and CYP11A1 gene expression and androgen biosynthesis in normal and PCOS theca cells propagated in long-term culture. The pathological characteristic of the PCOS ovary is that of multiple, small, growth arrested follicles. However, there have been no reports comparing cell growth or cell cycle control in normal and PCOS theca cells. We have identified differences in cell cycle regulation in normal and PCOS theca cells, and observed that metformin treatment of PCOS theca cells was observed to change PCOS and cell cycle progression to a normal phenotype. In Specific Aim 2, we will identify cell growth and cell cycle defects in PCOS theca cells and explore the signaling mechanism(s) involved in metformin's ability to correct these defects. The roles of LKB1, AMPK, and SIK1/2 in metformin-dependent cell growth and cell cycle progression, under normal physiological and hyperinsulinemic conditions, will be evaluated. We believe a clear understanding of mechanism(s) by which metformin signals to mediate inhibition of androgen biosynthesis and modulate cell growth in PCOS theca cells will help elucidate the pathophysiology of PCOS, and provide insight for the development of new therapeutic targets for PCOS. PUBLIC HEALTH RELEVANCE: Polycystic ovary syndrome (PCOS) is characterized by ovarian cysts, anovulation, hyperandrogenism, and endometrial hyperplasia, and affects 6-10% of reproductive age women, approximately 3-5 million women in the United States alone. Hyperandrogenism and anovulation in women with PCOS can be clinically treated with administration of the insulin sensitizing drug, metformin, a treatment for type 2 diabetes, however the mechanisms by which metformin directly modulates the ovary have not been examined. A rigorous evaluation of the mechanism(s) by which the metformin activates the LKB1/AMPK signaling pathway(s) to correct defects in androgen biosynthesis and cell growth in PCOS theca cells, will provide essential insight for the development of new therapeutic modalities for PCOS.